The goal of the collaborative research is to determine if the coupling of myocardial adenosine production to phosphoenergetic status is altered during hypoxia and ischemia, when normal energy balance is disturbed. He and co-workers observed that cytosolic AMP concentrations were completely dissociated from coronary venous adenosine release rate during myocardial underperfusion (1991), leading to the conclusion that there was an inhibition of 5'-nucleotidase (AMP -> adenosine) during ischemia. However, the conclusion of inhibition was not warranted, since a re-analysis of He's data using a mathematical model of adenosine transport and metabolism (Kroll et al., 1992) showed that the dissociation between AMP and adenosine release was probably due to more efficient adenosine salvage by capillary endothelial cells at low flow levels. In the collaborative project, total myocardial adenosine production during graded hypoxia and ischemia will be determined by measuring venous adenosine release in the presence of selective blockers of adenosine deaminase and adenosine kinase. With the enzymes blocked, there is reduced consumption of adenosine produced in the heart, and total myocardial adenosine production can be more accurately modeled by fitting the measurements of coronary venous adenosine. Cytosolic AMP concentrations will be estimated using 31-P NMR spectroscopy in the identical hearts used to measure adenosine production. A model of high energy phosphate kinetics (described in section D.II.A.2) will be used to estimate cytolic concentrations of AMP, based on modeling the NMR measurements of creatine phosphate and ATP. The planned experiments will provide the first direct in vivo estimates of precursor AMP concentrations and adenosine production rates, measured simultaneously during graded hypoxia and ischemia. The measurements will yield direct information on the relation of substrate level and enzyme velocity for the 5`-nucleotidase reaction. Thus, the in vivo kinetics of the major adenosine producing pathway can be assessed. Therefore, it will be possible to directly test the possibility that 5'-nucleotidase activity is altered during ischemia or hypoxia, and that there is differential regulation of the enzyme during hypoxia versus ischemia.